Lipidated glycoprotein particles and methods of use

ABSTRACT

Lipidated micro- or macroparticles are prepared by covalently linking a glycoprotein, typically collagen, with at least one lipid. An amino group in the glycoprotein is joined with a primary amine in the lipid. These particles can be used to encapsulate active ingredients, such as drugs.

FIELD OF THE INVENTION

The present invention relates to lipidated glycoprotein particles and tothe preparation and uses of microscopic and macroscopic drug deliverysystems such lipidated glycoprotein particles.

BACKGROUND OF THE INVENTION

Glycoproteins such as the fibrillar collagens, types I-III, are some ofthe main proteins in the extracellular matrix (ECM). Collagen isattached to specific cell-surface receptors that have the amino acidsequence Arg-Gly-Asp (Albelda & Buck, 1990; Dedhar, et al., 1987; andRuoslahti & Engvall, 1994); specifically this sequence known as the “RGDmotif” has been implicated as the cell attachment site of such ECMproteins as fibronectin, vitronectin, fibrinogen, and von Willebrandfactor, and is also present in type I collagen.

Because collagen is a biocompatible glycoprotein, there has beeninterest in developing collagenous drug carriers that can be loaded withdrugs and other bioactive agents.

There are two basic classes of drug carriers (Bangham, 1993; Benita &Levy, 1993; Gref et al., 1994; and Wu et al., 1994): (1) particulatesystems, such as cells, microspheres, viral envelopes, and liposomes and(2) non-particulate, usually soluble, systems consisting ofmacromolecules such as proteins or synthetic polymers.

Microscopic and macroscopic particulate carriers have several distinctadvantages over treatment with free drugs and non-particulate carriers.They can perform as sustained-release or controlled-release drug depots,thus contributing to improvement in drug efficacy and allowing reductionin the frequency of dosing. By protecting both the entrapped-drug andthe biological environment, these carriers reduce the risks of druginactivation and degradation. Since the pharmacokinetics of free drugrelease from the depots are different than from directly-administeredfree drug, these carriers have the potential to reduce toxicity andundesirable side effects.

Despite the advantages offered, the use of currently existing drugencapsulating particulate carriers has posed some challenges which haveyet to be fully overcome. For example, both macroparticulate andmicroparticulate drug delivery systems display limited targetingabilities; limited retention and stability in circulation; potentialtoxicity upon chronic administration; and an inability to extravasate.Numerous attempts have been made to bind substances such as antibodies,glycoproteins and lectins to particulate systems (e.g., liposomes,microspheres and others) in order to improve targeting ability.

Although bonding of these targeting agents to the particulate systemshas met with success, the resulting modified particulate systems havenot performed as hoped, particularly in vivo. Other difficulties arealso present. For example, for maximal effectiveness, antibodies shouldbe patient-specific and therefore add cost to the therapeutic regimen.

Further, not all binding between the targeting substance and the carrieris covalent. This type of bond is essential, as non-covalent bindingmight result in dissociation of the targeting substances from theparticulate system at the site of administration, due to competitionbetween the particulate system and the targeted components at the site.Upon such dissociation, the administered modified particulate systemwould likely revert to a conventional particulate system, therebydefeating the purpose of administration of the modified particulatesystem.

Therefore, there is a need in the art for a novel adhesive biopolymerthat can serve as a particulate carrier of drugs and other bioactiveagents. Such a biopolymer would be fully degradable and compatible inand with biological systems, unlike existing particulate carriers thathave non-biological components. Due to the use of biocompatible rawmaterials, this biopolymer would be nontoxic and nonimmunogenic, unlikesome of the existing carriers. (Toxicity and immunogenicity varies fromone carrier to another, but is on an acceptable level in those fewsystems approved for clinical use.).

The novel bioadhesive polymer serving as a particulate carrier shouldalso demonstrate high-efficiency entrapment independent of drug size upto and including proteins and genetic material, due to a “wraparound” or“induced-fit” nature. Existing particulate carriers demonstrate variableentrapment efficiencies ranging from low to high, with low efficiency ofhigh molecular weight entities. For intended use as a depot, it would bedesirable if the novel biopolymer exhibited exceptionally slow drugefflux, with a half-life in the range of 2-15 days. This would be incontrast to the high variability seen in conventional preparations, withdrug efflux ranging from fast to slow.

Additionally, it would be advantageous if the bioadhesive nature of theglycoprotein component would endow the system with the ability to adherewith high affinity to in vivo recognition sites and confer a measure ofactive targeting, in contrast with the conventional preparations. Inconventional preparations, further carrier modification is required toendow the systems with these properties, but is not always feasible andin some cases is counter-productive to production and to the intended invivo fate. It would be further desirable if the biopolymer comprised aglycoprotein, such as collagen, to which a lipid such asphosphatidylethanolamine were covalently linked.

SUMMARY OF THE INVENTION

It is an object of the present invention to overcome the deficiencies inthe prior art.

It is another object of the present invention to providemicroparticulate and macroparticulate drug/bioactive agent deliverysystems that utilize adhesive biopolymers with drug-entrappingcapability. When formulated with drugs or other bioactive agents, suchdelivery systems are expected to improve clinical outcomes, compared tothe same drugs administered in their free form. Composed of naturallyoccurring materials, which are both biocompatible and biodegradable, thebiopolymers of the present invention are used to encapsulateconventional drugs as well as other bioactive agents for a variety oftherapeutic and diagnostic purposes.

This novel drug delivery technology has many advantages over othercurrent systems. These include: high-efficiency encapsulation of largemacromolecules; exceptionally slow sustained-release; significantimprovement in the treatment of drug-resistant tumors; and noinflammatory response to in vivo implantation.

The present invention provides a drug delivery system for administrationof a bioactive agent to an animal comprising the bioadhesive polymericmacroparticles or microparticles of the present invention.

More particularly, the micro- or macroparticles comprise the reactionproduct of at least one amino-containing glycoprotein with at least onelipid having a primary amino group. In a preferred embodiment, theglycoprotein is collagen and the lipid is phosphatidylethanolamine.However, any glycoprotein may be used, provided it meets the followingbasic criteria. The glycoprotein must be sufficiently large in size(usually 100 kDa or larger), have sufficient free amino groups (at least5% of the amino acids should be lysine) and must possess an “RGDmotif”—the amino acids arginine, glycine and aspartic acid. Examples ofsuch glycoproteins are the collagens, fibronectin, vitronectin,fibrinogen, and von Willebrand factor.

Suitable lipids other than phosphatidylethanolamine, as would be wellappreciated by those of skill in the art, may be used, for example,phosphatidyl serine, phosphatidyl choline, phosphatidyl inositol,diphosphatidyl glycerol, phosphatidic acid, lysophosphatidic acid andacylphosphatidylethanolamine.

The microparticles of the present invention are vesicular-shaped,ranging in size from about 0.5-10 microns in diameter. Themacroparticles of the present invention are disk-shaped, ranging in sizefrom about 1-20 mm in diameter.

It is contemplated that an active ingredient would be encapsulatedwithin the micro- or macroparticles. Such active ingredients areselected from the group consisting of anti-infective agents,anti-neoplastic drugs, anti-viral agents, anti-microbial drugs,chemotherapeutic agents, anti-inflammatory agents, neuroleptics,fluorescent dyes, proteins, hormones, enzymes, cells, and nucleic acids.

The invention further provides a method of making a drug delivery systemin macroparticulate form, which comprises dissolving a glycoprotein;providing a reaction vessel in which a lipid is disposed in a thinlayer; buffering the lipid to a basic pH; admixing the dissolvedglycoprotein in the reaction vessel; adding a crosslinker; incubating aglycoprotein and lipid and cross-linker reaction mixture, withcontinuous shaking, for a period of time sufficient for glycoproteindisks to form; buffering a resultant lipidated glycoprotein to a neutralpH; separating unbound glycoprotein by centrifugation; and lyophilizinga resultant lipidated glycoprotein disk. For drugs and other bioactiveagents that are stable at basic pH, encapsulation may occur by addingthe drug when the crosslinker is added. In a preferred embodiment, theglycoprotein is collagen and the lipid is phosphatidylethanolamine. Thebasic pH is typically within the range from 8-10. Preferably, thecross-linker is glutaraldehyde.

The present invention further provides a method of making a drugdelivery system in microparticulate form, which comprises the same stepsas used for the macroparticulate drug delivery system, but with theadditional step of forming microparticles by means of the application ofmechanical forces to crush the glycoprotein disks into particles.

For those drugs that are not stable at a basic pH, the invention alsoprovides a method for making lipidated collagen disks or particleshaving an active ingredient entrapped therein, which comprisesreconstituting lyophilized collagen disks in water and adding a powderedactive ingredient, whereby the active ingredient is entrapped within thelipidated collagen disks.

The invention further provides a method for treating an animal sufferingfrom a pathological condition, which comprises administering to theanimal an effective amount of a bioactive agent encapsulated in micro-or macroparticles of a drug delivery system. The pathological conditionmay be, but is not limited to, cancer, bacterial infections includingosteomyelitis, fungal infections, viral infections, parasite infections,prion infections, or chronic conditions such as osteoarthritis,psychosis or hypertension.

In one embodiment, the pathological condition is cancer and thebioactive agent is an anticancer drug. In a preferred embodiment, thecancer is a cancer of a central nervous system of an animal,particularly a glioma. The cancer may also be colon adenocarcinoma. Inanother preferred embodiment, the cancer is a metastatic cancer. Theanti-cancer drug may be administered locally, or, in the case of acancer affecting the reticular endothelial system such as lymphomas orleukemias, it may be administered systemically.

Other pathological conditions are within the scope of the method oftreatment provided by the present invention. For example, in oneembodiment the pathological condition is a bacterial infection and thebioactive agent is an anti-bacterial drug. Treatment of such infectionsas (without limitation) fungal, viral, parasite and prion infections arecontemplated within the scope of the invention as well.

Chronic conditions such as osteoarthritis, psychosis or hypertension,where there are significant difficulties with patient compliance withdrug regimens are also contemplated as being within the scope of thepresent invention. In particular, the depot formulation lends itselfwell to such conditions where the patient must receive a constantlifelong dose of the appropriate drug.

Use in immunization programs is another application of the presentinvention. When vaccination programs must rely on patients returning for“booster” injections, they are often rendered ineffective due to patientnoncompliance. This is of concern particularly in developing countries.Long-acting, slow efflux of vaccine formulations encapsulated in thebiopolymers of the present invention would be useful in such a context.

Additionally, the present invention provides lipidated collagenparticles encapsulating a marker used in medical imaging. In a preferredembodiment, the marker is a radioactive isotope selected from the groupconsisting of ^(99m)Tc, ¹²⁷I, and ⁶⁷Gd. The lipidated collagen particlesmay also be formulated to encapsulate a fluorescent molecule. A methodfor diagnostic imaging a site in a patient using such particles is alsoprovided by the present invention.

Finally, the present invention also provides a method of gene deliveryand short term expression of an isolated nucleic acid segment in atarget cell or organ, which comprises administering to an animal in needthereof an effective amount of lipidated collagen particlesencapsulating nucleic acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scanning electron micrographic view into a collagen disk,showing the dense surface area and the porous nature of its interior.

FIG. 2 shows collagen particles with an average size distribution of0.5-10 μm, also using scanning electron microscopy.

FIG. 3 is confocal microscopy of PANC-1 (human pancreaticadenocarcimona) cells showing collagen particles entrapping protein(BSA-FITC). Cells were seeded into a 6 well plate. After 24 hours thecollagen-particles entrapping BSA-FITC were added to the cells andincubated for 1 hour at room temperature following three washings withPBS to separate the unbound particles from the cells. Then the cellsunderwent fixation for confocal microscopy and pictures were taken witha Zeiss camera.

FIG. 4 is confocal microscopy of C26 cells from mouse colonadenocarcimona showing collagen particles entrapping protein (BSA-FITC).Cells were seeded into a 6 well plate. After 24 hours thecollagen-particles entrapping BSA-FITC were added to the cells andincubated for 1 hour at room temperature following 3 washing with PBS toseparate the unbound particles from the cells. Then the cells underwentfixation for confocal microscopy and pictures were taken with a Zeisscamera.

FIG. 5 shows toxicity results for one concentration of drug-freecollagen particles (1 mg/ml), indicating that the particles alonedisplay no toxicity. In vitro toxicity was assayed by the MTT method(Wolff et al., 1999; and Nutt, 2000) on four types of cell lines. Thecell lines used were C6, a rat glioblastoma cell line; HT29, a humancolon adenocarcimona cell line; C26, a mouse colon adenocarcimona cellline; and PANC-1m a human pancreatic adenocarcimona cell line. Each baris an average of three independent experiments; each experimentconsisted of 60-64 repeats. The error bars represent the respectivestandard deviations.

FIGS. 6A-6C show results for three cell lines, C26 (FIG. 6A), HT29 (FIG.6B) and C26 (FIG. 6C), in terms of response to chemotherapeutic drugsencapsulated in collagen particles. The bars represent 60-64 repeats andthe error bars represent the standard deviation. Three asterisks (***)indicate p<0.001.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a drug delivery system for administrationof a bioactive agent to an animal comprising bioadhesive polymericmicro- or macroparticles. It is preferred that these particles comprisethe reaction product of at least one amino-containing glycoprotein withat least one lipid having a primary amino group. Optionally, yetpreferably, the glycoprotein is collagen and the lipid isphoshatidylethanolamine. The microparticles are generallyvesicular-shaped and preferably range in size from about 0.5 to 10microns in diameter. The macroparticles are generally disk-shaped andpreferably range in size from about 1-20 mm in diameter. In a preferredembodiment, the macroparticles are about 4 mm in diameter. In anotherpreferred embodiment, the macroparticles are about 10 mm in diameter.

Optionally, yet desirably, an active ingredient is encapsulated withinthe particles. Non-limiting examples of such active ingredients includeanti-infective agents, anti-inflammatory agents, anti-neoplastic drugs,anti-viral agents, anti-microbial drugs including anti-bacterial andanti-fungal drugs, chemotherapeutic agents, antipsychotic drugs, imagingagents/markers such as fluorescent dyes, proteins, hormones, enzymes,cells, and nucleic acids.

The invention further provides a method of making a drug delivery systemin macroparticulate form. The method comprises an initial step ofdissolving a glycoprotein. Such a dissolving step may be accomplished,for example, by pre-incubation in mildly acidic pH overnight at 4° C. Ina preferred embodiment, the glycoprotein is collagen.

At a time which may be simultaneous with the initial glycoproteindissolving step, a reaction vessel in which a lipid is disposed in athin layer on the vessel bottom and walls is provided. This dispositionmay be accomplished by dissolving the lipid in an organic solvent andevaporating to dryness under low pressure in a rotary evaporator. In apreferred embodiment, the lipid is phosphatidylethanolamine. Once thedisposition of the lipid is achieved, the reaction mixture comprisingthe lipid in the reaction vessel is to be buffered to a basic pH.Optionally, the basic pH is in the range of 8-10.

The dissolved glycoprotein is then admixed into the reaction vessel. Acrosslinker, such as glutaraldehyde, is then added. Bioactive agentsthat do not lose their activity at a basic pH may be added in this stepas well.

The resultant reaction mixture is then incubated, with continuousshaking, under time and temperature conditions sufficient for theglycoprotein disks to form, for example, overnight at 4° C. Theresultant lipidated glycoprotein is buffered to a neutral pH. Other ionsand water-soluble additives may then be added to the mix according toneed in order to elevate the ionic strength to physiological levels withions or salts present in biological fluids such as: NaCl, KCl, Ca²⁺ andMg²⁺. Any unbound glycoproteins are subsequently separated out bycentrifugation at progressively higher g forces. The lipidatedglycoprotein is then lyophilized, resulting in stable disks.

The present invention also provides a method of making a drug deliverysystem in microparticulate form. The method comprises identical steps asthe method for making a drug delivery system in macroparticulate form,but with an additional step of forming stable microparticles throughapplying mechanical forces sufficient to crush the macroparticle disksinto particles.

The present invention further provides a method for making lipidatedglycoprotein macro- or microparticles having an active ingrediententrapped therein, comprising reconstituting lyophilized collagen disksin water, and adding a powdered active ingredient, whereby the activeingredient is entrapped within the lipidated collagen disks. This methodis suitable for use when the active ingredient of interest would loseits biological activity at a basic pH and therefore cannot be addedduring the micro- or macroparticle formulation process. This methodcomprises reconstituting lyophilized lipidated collagen particles inwater, and adding a powdered active ingredient, whereby the activeingredient is entrapped within the lipidated collagen particles.

A method for treating an animal suffering from a pathological conditionis additionally furnished by the present invention, comprisingadministering to the animal in need thereof an effective amount of abioactive agent encapsulated in the micro- or macroparticles of the drugdelivery system of the instant invention. The pathological condition maybe, but is not limited to osteoarthritis, cancer, bacterial infectionsincluding osteomyelitis, fungal infections, viral infections, parasiteinfections, psychoses and prion infections.

Exceptionally resistant cancers such as gliomas and colonadenocarcinomas are particularly suited for in situ therapies utilizingthe treatment method of the present invention. Cancers in general areuseful targets of the glycoprotein biopolymers because tumors are areasvery rich in cell surface receptors, such as integrins, that bindglycoproteins such as collagen. The vicinity of a tumor is also highlyenriched in extracellular matrix (ECM) proteins. Indeed, it is proposedthat by blocking the receptors with the empty particles of the presentinvention (particles free of any therapeutic agent), inhibition ofmetastasis occurs because the tumor cannot use the receptors in itsmetastatic process.

Because the invention contemplates the use of fluorescent andradioactive markers for diagnostic and therapeutic use, the inventionfurther provides lipidated collagen particles encapsulating a markerused in medical imaging. In a preferred embodiment, the marker may be aradioactive isotope such as ^(99m)Tc, ¹²⁷I, and ⁶⁷Gd. Lipidated collagenparticles encapsulating a fluorescent molecule are also contemplated tobe within the scope of the invention. The present invention additionallyfurnishes a method for diagnostic imaging a site in a patient using theparticles.

The present invention also furnishes a method of gene delivery and shortterm expression of an isolated nucleic acid segment to a target regioncomprising administering to an animal in need thereof an effectiveamount of lipidated collagen particles encapsulating nucleic acids.Applications of the gene therapy include, without limitation, treatmentof cancer and other molecular disorders. As discussed above, cancers ingeneral are useful targets of the glycoprotein biopolymers because thesepolymers home to sites rich in those cell surface receptors, such asintegrins, that bind glycoproteins such as collagen. Therefore, tumorsmay be targeted by such polymers for therapeutic purposes.

The present invention further provides an improvement to methods fortreating a given indication with a drug that is effective for treatingthe indication. The improvement results from the drug being administeredencapsulated in lipidated glycoprotein microparticles or macroparticles,also referred to as “collagomers”. For instance, in a pathologicalcondition such as osteomyelitis (bone infection), antibiotics, e.g.,anti-bacterials such as cephalosporins, can be locally administered.Likewise, to treat osteoarthritis, anti-inflammatory drugs, such asacetaminophen, COX-2 inhibitors (CELEBREX, ROFECOXIB, etc.), andnon-steroidal anti-inflammatory drugs (NSAIDs) including aspirin,ibuprofen, naproxen, diclofenac, and ketoprofen can be administeredlocally by encapsulating such anti-inflammatory drugs in the collagomersof the present invention.

As used herein, the term “drug” is identical to that employed in the26^(th) Edition of Stedman's Medical Dictionary, viz., “[a][t]herapeutic agent; any substance, other than food, used in theprevention, diagnosis, alleviation, treatment, or cure of disease.”

In addition, for the purposes of the present invention, a drug may beany substance that affects the activity of a specific cell, bodily organor function. A drug may be an organic or inorganic chemical, abiomaterial, etc. Any chemical entity of varying molecular size (bothsmall and large) exhibiting a therapeutic effect in animals and humansand/or used in the diagnosis of any pathological condition, includingsubstances useful for medical imaging such as fluorescent dyes andradioactive isotopes fits the above definition.

Active agents that can be delivered according to the present inventioninclude inorganic and organic drugs without limitation and include drugsthat act on the peripheral nerves, adrenergic receptors, cholinergicreceptors, nervous system, skeletal muscles, cardiovascular system,smooth muscles, blood circulatory system, synaptic sites, neuro-effectorjunctional sites, endocrine system, hormone systems, immune system,reproductive system, skeletal system, autocoid systems, alimentary andexcretory systems, histamine systems and the like.

An active drug that can be delivered for acting on these recipients canbe water soluble or water insoluble/poorly soluble and includes, but isnot limited to, anticonvulsants, analgesics, anti-Parkinson's,anti-inflammatories, calcium antagonists, anesthetics, antimicrobials,antihypertensives, antihistamines, antipyretics, alpha-adrenergicagonists, antipsychotics, alpha-blockers, biocides, bronchial dilators,beta-adrenergic blocking drugs, contraceptives, cardiovascular drugs,calcium channel inhibitors, antidepressants, diagnostics, diuretics,electrolytes, enzymes, hypnotics, hormones, hypoglycemics,hyperglycemics, muscle contractants, muscle relaxants, neoplastics,glycoproteins, nucleoproteins, lipoproteins, opthalmics, sedatives,steroids, sympathomimetics, tranquilizers, vaccines, vitamins,nonsteroidal anti-inflammatory drugs, angiotensin converting enzymes,polynucleotides, polypeptides, polysaccharides, and the like.

Of particular interest is the possibility of applying the drug deliverytechnology for use as a depot in administering drugs for chroniclong-term use and for vaccination. Also, leaving a depot in placefollowing surgery may be advantageous. In the former instance, there aresignificant problems with patient compliance when drugs for conditionssuch as hypertension or mental illnesses are prescribed. Regimes such asdepot administration would be beneficial in eliminating this problem.Also, in many instances, e.g., developing nations, etc., vaccinationprograms are rendered ineffective as patients do not return for their“booster” immunizations. A long acting depot preparation could solvethis problem.

This technology is different from that of the drug delivery technologyinvented by D. Peer and R. Margalit, denoted lipidatedglycosaminoglycans (or gagomers) (PCT International Application No.PCT/US02/25178 and U.S. Patent Application PublicationU.S.2004/0241248). There are several key differences between thecollagen-based particles of the present technology and the hyaluronicacid-based particles of the other technology.

The first key difference involves scale: the collagomers are on amacro—(mm) and micro—(μm) scale, whereas the gagomers are on amicro—(μm) and nano—(nm) scale. Another key difference lies in thenature of the biological recognition sites. The collagomers recognizecollagen receptors and collagen-binding proteins with the RGD motif. Bycontrast, the gagomers recognize hyaluronic acid receptors andhyaluronic acid-binding proteins. Additionally, while the collagomers,due to their size, are most suitable for local administration and asimplants, the gagomers may be used for both systemic and localadministration. Finally, the collagomers display extremely slow releasetimes, with a typical half-life from disks >15 days, while the gagomershave a slow release time, with a typical half-life 1-3 days.

The collagomers may be formulated to entrap therapeutic compositions fordrug or gene therapy, or may be empty, for use as drug carriers. Resultsfrom animal studies suggest that empty collagomers may even have sometherapeutic utility in treating cancer, especially metastatic cancer, byblocking the cell surface receptors to which the metastasizing tumorcells would bind.

Depending on the intended mode of administration, the compositions usedmay be in the form of solid, semi-solid or liquid dosage forms suchpharmaceutical compositions will include the collagomer construct asdescribed and a pharmaceutical acceptable excipient, and, optionally,may include other medicinal agents, pharmaceutical agents, carriers,adjuvants, etc. It is preferred that the pharmaceutically acceptablecarrier is chemically inert to the active compounds and have nodetrimental side effects or toxicity under the conditions of use.

The choice of carrier is determined partly by the particular activeingredient, as well as by the particular method used to administer thecomposition. Accordingly, there are a wide variety of suitableformulations of the pharmaceutical compositions of the presentinvention.

Suitable excipients are, in particular, fillers such as saccharides, forexample, lactose or sucrose, mannitol or sorbitol, cellulosepreparations and/or calcium phosphates, for example, tricalciumphosphate or calcium hydrogen phosphate, as well as binders such asstarch paste using, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, tragacanth, methylcellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/orpolyvinyl pyrrolidine. Suitable formulations can be found in Remington'sPharmaceutical Sciences, 16^(th) and 18^(th) Eds., Mack Publishing,Easton, Pa. (1980 and 1990), and Induction to Pharmaceutical DosageForms, 4^(th) Edition, Lea & Febiger, Philadelphia (1985), each of whichis incorporated herein by reference.

Pharmaceutical compositions using the collagomers according to thepresent invention can be administered by any convenient route, includingparenteral, e.g., subcutaneous, intravenous, intranasal, topical,intramuscular, intraperitoneal, etc. Alternatively or concomitantly,administration may be by the oral route.

Parenteral administration is usually characterized by injection, mosttypically subcutaneous, intramuscular or intravenous. Parenteraladministration can be by bolus injection or by gradual perfusion overtime.

Means by which the collagomers may be administered are encompassed underthe rubric of drug delivery devices. The term “drug delivery device”refers to any means for containing and releasing a drug wherein the drugis released into a subject. Drug delivery devices are split into fivemajor groups: inhaled, oral, transdermal, parenteral and suppository.Inhaled devices include gaseous, misting, emulsifying and nebulizingbronchial (including nasal) inhalers; oral includes mostly pills;whereas transdermal includes mostly patches. Parenteral includes twosub-groups: injectable and non-injectable devices. Non-injectabledevices are generally referred to as “implants” or “non-injectableimplants” and include, e.g., pumps and solid biodegradable polymers.Injectable devices are split into bolus injections, that are injectedand dissipate, releasing a drug all at once, and depots, that remaindiscrete at the site of injection, releasing drug over time. Many drugdelivery devices are described in Encyclopedia of Controlled DrugDelivery (1999), Edith Mathiowitz (Ed.), John Wiley & Sons, Inc.

A preferred embodiment of the drug delivery device can be a depot. Asnoted supra, depots are injectable drug delivery devices that maycomprise polymeric and/or non-polymeric materials, and are provided inliquid, or semi-solid forms that release drug over time. “Depot” means alocalized site in the body containing concentrated active agents ordrugs. Examples of formulations that form depots are gels, implants,microspheres, matrices, particles, oils, liquid polymers andnon-polymers, etc. A most preferred embodiment consists of theformulation in the form of a depot comprising microspheres.

EXAMPLE 1 Synthesis of Drug-Entrapping Collagomers

Two basic types of collagomers were synthesized: disk-shapedmacroparticles and vesicular-shaped microparticles.

Particle Preparation

Among the drugs/bioactive agents which may be entrapped in thecollagomer of the present invention are: the fluorescent dye moleculefluorescein (Flu); the anti-microbial chloramphenicol (CAM), theantineoplastics doxorubicin (DOX), Mitomycin C (MMC) and vinblastine(VIN); bovine serum albumin (BSA) conjugated to the fluorescent markerfluorescein isothiocyanate (FITC) (BSA-FITC); the antiviral proteininterferon 2α (INFα) and plasmid DNA (pMC1403 from E coli).

A process for creating glycoprotein disks or particles by covalentlybinding a lipid having a primary amino group to an amino containingglycoprotein comprises the steps of:

(a) Dissolving a glycoprotein by pre-incubation in mildly acidic pHovernight at 4° C.;

(b) Providing a reaction vessel wherein a lipid is disposed in a thinlayer on the vessel bottom and walls, e.g., by dissolving the lipid inan organic solvent and evaporating to dryness under low pressure in arotary evaporator;

(c) Buffering the reaction mixture of the lipid to a basic pH (8-10);

(d) Admixing the dissolved glycoprotein to the reaction vessel andadding a crosslinker such as glutaraldehyde (GAD) (at this point, drugswhich do not lose their biological activity at basic pH levels may beadded);

(e) Incubating the buffered reaction mixture, with continuous shaking,for a period of time sufficient for the collagen disks or particles toform (such as overnight at 4° C.);

(f) Buffering the lipidated collagen to neutral pH and adding other ionsand water-soluble additives (such as NaCl, KCl, Ca²⁺ and Mg²⁺) accordingto need;

(g) Separating the unbound glycoproteins by centrifugation atprogressively higher g forces;

(h) Lyophilizing the lipidated glycoprotein, which results in a stabledisk; and

(i) Forming, when desired, stable microparticles, through applyingmechanical forces to crush disks into particles.

Entrapment of Bioactive Agents

There are two ways by which bioactive agents such as drugs can beentrapped in the collagomers:

1. (a) Dissolving the drug/bioactive agent of interest in ion-free purewater; and

(b) Reconstituting the carrier from the lyophilized dry powder obtainedin step I(h) above (when no drug was added by step I (d)) byre-hydrating the powder with solution from 1(a) above; or

2. For drugs that are stable in basic pH—dissolving the drug/bioactiveagent of interest in step I(d) above.

EXAMPLE 2 Structural Properties of Disks and Particles

Investigation of the structural properties of the macroparticulate disksand microparticulate vesicles were focused on the collagomer itself andon chemical bonding within it. Scanning electron microscopy (SEM) wasperformed.

FIG. 1 is an EM cross-sectional view of a disk, showing a dense surfacearea and the porous nature of its interior. The disk sizes were measuredby electronic calipers and typical size distribution were between 1-20mm in diameter. The disks were made by the process of Example 1 usingtype I collagen as the glycoprotein and phosphatidylethanolamine as thelipid and stopping the reaction after step (h).

FIG. 2 shows microparticles with an average size distribution of 0.5-10μm in diameter. Size distribution was further confirmed by an ALV-NIBSparticle size analyzer (ALV-GmbH, Langen/Germany). The microparticleswere made by treating the disks of FIG. 1 according to step (i) of theparticle preparation process, where disks were crushed by mechanicalforces into microparticles.

EXAMPLE 3 Physicochemical Characterization

Efficiency of entrapment of drugs/bioactive agents in the collagen disksor particles, and kinetics of drug efflux for small molecular weightdrugs was determined utilizing absorption spectra (mostly in an ELISAplate reader), fluorescence emission spectra (mostly in a fluorescenceplate reader), and HPLC. Half-life data was processed according to atheoretical framework previously developed for liposomes (Margalit etal., 1991), incorporated herein in its entirety) which yields the rateconstant for drug efflux. Typical results of the efficiency ofentrapment are listed in Tables 1 and 2, for the particles and disks,respectively.

TABLE 1 Collagen particles (0.5-10 μm in diameter) Low Molecular weightDrugs Drug % Entrapment Slow release (half-life) DOX 71.9 ± 0.4 13 daysMMC 67.3 ± 2.1  6 days CAM 94.1 ± 0.8 15 days Flu 95.6 ± 2.8  9 daysHigh Molecular weight Drugs Drug % Entrapment BSA 82.0 ± 2.3 Insulin91.2 ± 0.7 Interferon alpha 88.2 ± 5.1 DNA 80.7 ± 2.1

TABLE 2 Collagen Disks (1-20 mm in a diameter) Drug % Entrapment Slowrelease (half-life) Low Molecular weight Drugs DOX 78.4 ± 2.4 15 daysMMC 80.2 ± 1.0 12 days CAM 89.5 ± 1.4 19 days High molecular weightdrugs BSA 87.5 ± 1.7 17 days Insulin 89.4 ± 2.3 21 days Interferon alpha93.6 ± 1.3 24 days DNA 85.1 ± 4.3

The results shown in these tables demonstrate that both small and largedrugs can be entrapped in collagen disks and particles with highefficiency. Efficiency of entrapment is exceptionally high for the largemolecules compared to other particulate carriers such as liposomes andmicrospheres. Typical examples of the kinetics of drug efflux from thedisks and from the particles is shown in Tables 1 and 2 for small aswell as large molecules. In all cases the systems perform assustained-release drug depots, as desired, with most half-lives on theorder of 6-24 days, depending on the drug itself and the degradationrate of the disks and particles.

EXAMPLE 4 Confocal Microscopy

BSA-FITC was entrapped in the particles. Using confocal microscopy itwas found, as shown in FIGS. 3 and 4, that the particles attach to theadenocarcinoma cells but do not enter. The two cell lines are PANC-1(human pancreatic adenocarcimona) and C26 (mouse colon adenocarcimona),for FIGS. 3 and 4, respectively.

EXAMPLE 5 Biological Activity: In Vitro Toxicity Studies

Drug-free collagen particles were tested for toxicity in cell cultures.Four cell lines were tested: the rat glioma line C6; the human colonadenocarcimona HT29 cell line; the human pancreatic adenocarcimonaPANC-1 cell line; and the mouse colon adenocarcimona C26 line. In allcases, the collagen particles were found to have no toxicity over the100-fold concentration range of 0.02 to 2 mg/ml polymer. FIG. 5demonstrates those findings for one concentration of drug-free collagenparticles (1 mg/ml).

Particles were also tested for their toxicity to noncancerous celllines, the NIH 3T3 mouse fibroblast cell line (results not shown). Notoxicity was observed.

EXAMPLE 6 Biological Activity: Treatment of Multi-Drug Resistant CellLines Originating From Tumors

Poor response to chemotherapeutic drugs due to drug resistance andclinically challenging location are among the major causes for thefrequent failures in treatment of brain and colon tumors, especiallygliomas (Wolff et al., 1999; and Nutt, 2000), and colon adenocarcinoma.The poor drug response is due in part to lack of access, and in part toinherent multidrug resistance (MDR) (Gottesman, et al., 1995; and Larsenet al., 2000). In brain tumors, MDR is an impediment even in cases whereaccess to the tumor has been provided, for example by localadministration or by provision of a drug depot at the end of a surgicalprocedure. Similar difficulties are observed in the case of colonadenocarcinoma.

In this prevalent drug resistance mechanism, which appears in both anacquired and inherent mode, the drugs do not lose their intrinsic toxicactivity, nor have the resistant cells found a way to metabolize thedrugs into nontoxic entities. Rather, any drug that enters the cellthrough passive diffusion across the cell membrane is actively pumpedout, reducing intracellular levels below their lethal threshold.

The glioma C6 line, which displays inherent MDR, and the two colonadenocarcimona cell lines in current use (HT29—human and C26—mouse)served as model systems for testing whether treatment with collagenparticles entrapping a chemotherapeutic drug (doxorubicin, DOX) wouldoffer any advantage over a similar treatment with the free drug.

Cells were seeded onto 96 well plates, and the experiment was initiatedat semi-confluency, usually 24 hours post seeding. For the test system,cells were given a selected dose of the drug of choice, which wasentrapped in a collagen particle formulation (washed of excess free drugprior to use). The controls were given the same dose of free drug, and adose of drug-free collagen particles at a dose similar to that of thetest system. Cell survival was determined 24-28 hours post-treatment,using the MTT assay (Nutt, et al. 2000; and Larsen et al., 2000).

Results for three cell lines (C6, HT29 and C26) are shown in FIGS. 6A-6Cin three data sets. The data for the free particles (left-most bar ineach of the three data sets) is an additional confirmation that thecollagen particles are nontoxic. The responses to free drug (DOX) shownin the middle bar of each data set, are typical for the inherent form ofMDR.

Replacing the free drug with the same dose ofcollagen-particles-entrapped drug generated a dramatic difference, asseen by the right-hand bar in each data set. For each of the three celllines, the novel formulation generates a 3-4 fold increase in celldeath, compared to the corresponding free drug. Because of the non-toxicnature of the free particle, it is assumed that these resultsdemonstrate the tumoricidal properties of the collagomers.

In theory, all that is required to overcome MDR is to find a way toelevate intracellular doses of a chemotherapeutic drug above the lethalthreshold. The traditional approach has been to reduce the pumping byusing reversal agents known as chemosensitizers. While several suchagents have been identified—most prominent among them verapamil—they arenot yet in clinical use for the treatment of cancer. This is due to thefact that these chemosensitizers cannot be used in the clinic, sincedose levels that cause unacceptable adverse effects and toxicity wouldbe required. It is important to note that such treatment would requirecareful orchestration as both active entities, the chemotherapeutic drugand the chemosensitizer, must reach the target together. This is not asimple matter in clinical practice.

However, another way to elevate the intracellular drug dose is toincrease influx of drug, in terms of magnitude and duration thereof. Itis suggested that the outstanding increase in drug response generated bythe drug-entrapping particles occurs by increasing influx. Thebioadhesive nature of the collagen-particles positions them as drugdepots bound to the cell membrane, which should increase the magnitudeof the electrochemical gradient of the drug across the cell membrane(compared to free drug), and the time span during which drug entryoccurs. Efficacious cancer treatment could therefore require only oneentity, the drug-collagomer formulation, which should be an advantageover the drug and chemosensitizer combination.

It is suggested that the potential of overcoming MDR that has emergedfrom the present studies is not restricted to the C6, HT29 and C26 celllines, but is general to other MDR cases, both inherent and acquired.Moreover, these new formulations may also benefit non-resistant tumors,by allowing successful treatment with significantly lower drug doses.

EXAMPLE 7 In Vivo Toxicity

The question of whether these novel carriers are toxic in vivo wasinvestigated in two strains of mice and in one strain of rats. Studieswere performed separately for the collagen disks and for the particles.

Experiment 1

Collagen disks were implanted in the right flanks of four C57BL/6 mice.The size of each collagen disk was 4 mm in diameter. The mice wereobserved for 2 months and assessed for clinical indication ofinflammatory reactions at the implanted site. No inflammatory reactionwas observed in those mice.

Experiment 2

Larger collagen disks (an average of 10 mm in diameter) were implantedin the right flanks of two pigmented rats. As in the case of the miceabove, there were no clinical indications of any inflammatory reactionat the site of the operation, as observed over a period of two months.At the end of two months, one rat was sacrificed, and an incision wasmade at the implant location, in order to view the internal placement ofthe disk. The disk was found adhered to muscle tissue and was almost atits original size. The internal tissues were clear of inflammation, andthere was no capsule build-up around the implanted disk.

Experiment 3

Collagen particles were administrated intravenously into the tail veinof four BALB/c mice. No toxicity was observed over 30 days.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the inventions following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

All references cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedU.S. or foreign patents, or any other references, are entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited references. Additionally, the entirecontents of the references cited within the references cited herein arealso entirely incorporated by reference.

Reference to known method steps, conventional methods steps, knownmethods or conventional methods is not in any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

Thus the expressions “means to . . . ” and “means for . . . ”, or anymethod step language, as may be found in the specification above and/orin the claims below, followed by a functional statement, are intended todefine and cover whatever structural, physical, chemical or electricalelement or structure, or whatever method step, which may now or in thefuture exist which carries out the recited function, whether or notprecisely equivalent to the embodiment or embodiments disclosed in thespecification above, i.e., other means or steps for carrying out thesame functions can be used; and it is intended that such expressions begiven their broadest interpretation.

REFERENCES

-   Albelda, S M, and Buck, C A., FASEB J. 4:2868-2880 (1990)-   Bangham, A. D., Chem. Phys. Lipids, 64:275-285 (1993)-   Benita S. and Levy M Y, J. Pharmaceutical, Sciences 82:1069-1079    (1993)-   Dedhar, S., Ruoslahti, E., and Pierschbacher, M D., J Cell Biol,    104:585-593 (1987)-   Gottesman, M M, Hrycyna, C A, Schoenlein, P V, Germann, U A and    Pastan, I., Annu. Rev. Genet. 29:607-649 (1995)-   Gref, R., Minamitake, Y., Peracchia, M T, Trubetskov, V, Torchilin,    V and Langer R., Science 263:1600-1603 (1994)-   Larsen, A K, Escargueil, A E and Skladanowski, A., Pharmacol. Ther.    85:217-229 (2000)-   Margalit R, Alon R, Lindenberg M, Rubin I, Roseman T J, and Wood, R    W, J. Controlled Release, 17:285-296 (1991)-   Nutt C L, Cancer research, 65:4812-4818 (2000)-   Ruoslahti, E., and Engvall E., editors. Methods in Enzymology vol.    245, Extracellular matrix components. Academic press, San Diego,    (1994)-   Wolff J E, Trilling T, Molenkamp G, Egeler R M, Jurgens H, J. Cancer    Res. Clin. Oncol. 125, 481-486 (1999)-   Wu et al., J. Biomed. Mater. Res. 28: 387-395 (1994)

What is claimed is:
 1. A method for treating a patient suffering fromosteoarthritis, comprising administering to the patient in need thereofan effective amount of a lipidated glycoprotein microparticle ormacroparticle encapsulating diclofenac, wherein the lipidatedglycoprotein microparticle or macroparticle has a solid porous interiorand comprises the reaction product of at least one amino-containingglycoprotein covalently bound with at least one lipid having a primaryamino group.
 2. The method of claim 1, wherein the lipid in thelipidated glycoprotein microparticle or macroparticle isphosphatidylethanolamine.
 3. The method of claim 1, wherein theglycoprotein in the lipidated glycoprotein microparticle ormacroparticle is collagen.
 4. The method of claim 1, wherein thelipidated glycoprotein microparticle or macroparticle consists of amicroparticle ranging in size from about 0.5 to about 10 microns indiameter.
 5. The method of claim 1, wherein the lipidated glycoproteinmicroparticle or macroparticle consists of a macroparticle ranging insize from about 1 mm to about 20 mm in diameter.
 6. The method of claim5, wherein the macroparticle is about 4 mm in diameter.
 7. The method ofclaim 5, wherein the macroparticle is about 10 mm in diameter.
 8. Themethod of claim 1, wherein said at least one amino-containingglycoprotein comprises an RGD motif of amino acid sequence Arg-Gly-Asp.9. The method of claim 1, wherein said at least one amino-containingglycoprotein is 100 kDa or larger and has at least 5% of its amino acidsas lysine.